CN101537189A - Aptamer and new use of derivative thereof - Google Patents
Aptamer and new use of derivative thereof Download PDFInfo
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- CN101537189A CN101537189A CN200910083080A CN200910083080A CN101537189A CN 101537189 A CN101537189 A CN 101537189A CN 200910083080 A CN200910083080 A CN 200910083080A CN 200910083080 A CN200910083080 A CN 200910083080A CN 101537189 A CN101537189 A CN 101537189A
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Abstract
The invention discloses an aptamer and a new use of derivative thereof. The new use provided by the invention is application of aptamer and the derivative thereof in preparation of target drugs. The invention further protects the target drugs obtained by coupling the drugs with the aptamer or the derivative thereof. The invention has the advantages of screening and preparing specific aptamer or derivative thereof under the condition of not knowing the molecular transformation of the target cell surface related to the disease, and can simultaneously obtain multiple aptamers or derivatives thereof of molecular related to the cell surface cell; the specificity of the obtained aptamer or derivative thereof is strongly combined with the disease cell; the aptamer or the derivative thereof can be chemically synthesized in large scale and can be easily connected with the drug molecules and has low cost; the molecular weight of the aptamer or the derivative thereof is relatively small without immunologic competence and toxicity; the aptamer or the derivative thereof has good stability; and the formed target drugs are stable. The invention has very high application value.
Description
Technical field
The present invention relates to the new purposes of aptamer and derivant thereof.
Background technology
Traditional tumor chemotherapeutic drug often also kills and wounds normal cell owing to not strong to the specificity of tumor cell in the kill tumor cell, cause strong toxic and side effects.The tumor cell target administration is to improve the effective way that the oncotherapy effect reduces toxic and side effects.With drug coupling is the main method of target administration in the tumor cell specific aglucon.For example medicine is covalently bonded on the monoclonal antibody of cell surface tumor markers, the monoclonal antibody of these coupling drugs energy selective binding tumor tissues, thus the effect of raising antitumor drug reduces toxic and side effects.The target therapeutic agent of a plurality of different mechanisms of action share to obtain better therapeutic effect.A lot of these class medicines have all been obtained good effect in preclinical test, the clinical experiment (Carter, the P.J. that are being correlated with at present; Senter, P.D.Cancer J.2008,14,154-169.Chari, R.V.J.Acc.Chem.Res.2008,41,98-107.).For example humanized CD33 antibody-Gary mycin conjugate Mylotarg that stops has been approved for the treatment of acute myeloid lineage leukemia (AML).The antibody sources that is used for the drug targeting conveying is in the monoclonal cell of animal or fusion, it is the product of nature, its preparation is loaded down with trivial details, poor stability, molecular weight greatly, easily cause immunoreation and be difficult to carry out chemical modification and transformation etc., therefore cause the antibody coupling matter poor stability, the cost height has limited its extensive use.Seek new specific recognition molecule and replace the important development direction that antibody is the targeted drug treatment.
A class novel identification molecule-aptamer (Aptamer, aptamers, adaptive son) obtains people's attention gradually with its superior evident characteristics in recent years.Aptamer is one section single stranded nucleic acid molecule (comprises DNA, RNA, modification after nucleic acid molecules).They are folded to form unique three-D space structure by intramolecularly base stacking, hydrophobic interaction, hydrogen bond and electrostatic interaction, thereby combine with the target molecule specificity (Breaker R R, Nature2004,432:838-845).Aptamer is similar to monoclonal antibody to specificity to the bonded affinity of target molecule, even stronger.Compare with antibody, aptamer has lower molecular weight (15-50bases; 4-15kD), fast tissue permeability, do not have immunogenicity and toxicity.In a single day aptamer screens, and just can synthesize by the solid state chemistry of automatization to prepare, and need not the process and the follow-up loaded down with trivial details separation and purification process of immune animal or cell culture, and price reduces greatly.Aptamer is easy to carry out various molecules on structure of modification and the labelling, as (Nutiu R ﹠amp such as radioelement, fluorescent dyes; Li Y, Angew Chem Int Ed Engl 2005,44:1061-1065; Liu, J.W.﹠amp; Lu, Y.Angew Chem Int Ed Engl 2006,45:90-94).Aptamer stability is high, can reversible degeneration and renaturation, storage and transport at normal temperatures.
Summary of the invention
The new purposes that the purpose of this invention is to provide aptamer and derivant thereof.
The invention provides the application in the preparation targeted drug of aptamer and/or nucleic acid aptamer derivative.
Described nucleic acid aptamer derivative can be following a), b) or c):
A) skeleton with aptamer partly or entirely transform phosphorothioate backbone as or the sugar ring on the part or all of skeleton is carried out 2 '-F and 2 '-NH
2Modify and wait the nucleic acid aptamer derivative that obtains;
B) partly or entirely transform aptamer as nucleic acid aptamer derivative that peptide nucleic acid(PNA) obtains;
C) an above nucleotide in the aptamer is replaced with lock nucleic acid (Locked Nucleic Acid, LNA) and/or the nucleic acid aptamer derivative that obtains of link molecule; Described link molecule is more than one six Polyethylene Glycol-1-phosphoric acid.
The structural formula of six Polyethylene Glycol-1-phosphoric acid is suc as formula (I):
Shown in (I), the skeleton of six Polyethylene Glycol-1-phosphoric acid is the polymer of 6 ethylene glycol, and the oxygen of skeleton one end connects an electronegative phosphate group.When substituting nucleotide in the aptamer with six Polyethylene Glycol-1-phosphoric acid, the oxygen by the skeleton two ends is connected with the nucleotide at two ends.
Described aptamer be can with bonded one section single stranded nucleic acid molecule of disease association molecular specific or modified nucleic acid molecules.
Described targeted drug can be the medicine of treatment tumor, the medicine that treatment is infected or the medicine for the treatment of inflammation.
The medicine of described treatment tumor specifically can be the kill tumor cell and/or suppresses the medicine of tumor cell proliferation.
Described aptamer can be the DNA that contains core sequence.
Described aptamer also can contain flanking sequence.Described flanking sequence can be 5 ' distolateral wing sequence and/or 3 ' distolateral wing sequence.
Described tumor cell specifically can be the leukaemia.
When described tumor cell when the leukaemia: described core sequence can be in sequence 1, sequence 2, sequence 3, sequence 4, sequence 5, sequence 6, sequence 7, sequence 8, sequence 9, sequence 10 or the sequence 11 in the sequence table DNA sequence shown in any ten above continuous nucleotides; Described 5 ' distolateral wing sequence can be the DNA sequence shown in any three above continuous nucleotides in the sequence 12 in the sequence table; Described 3 ' distolateral wing sequence can be the DNA sequence shown in any three above continuous nucleotides in the sequence 13 in the sequence table.
When described tumor cell was the leukaemia: described aptamer was specific as follows:
5 '-NTCTAACTGCTGCGCCGCCGGGAAAATACTGTACGGTTAGA-3 ' (sequence 14 of sequence table);
5 ' end of above-mentioned sequence is any nucleotide, is used for labelling usefulness, as A.
When described tumor cell was the leukaemia: described nucleic acid aptamer derivative was specific as follows:
5′-N
LT
LC
LT
LAACTGCTGCGCCGCCGGGpegGTACGGTTA
LG
LA
L-3′；
Above-mentioned nucleic acid aptamer derivative is that 3 nucleotide with 4 nucleotide of 5 ' end of the aptamer shown in the sequence 14 of sequence table and 3 ' end replace with corresponding lock nucleic acid unit, will replace with that described link molecule obtains from 5 ' terminal the 23rd to 30 nucleotide.
Described link molecule specifically can be 2 to 5 six Polyethylene Glycol-1-phosphoric acid, specifically can be two six Polyethylene Glycol-1-phosphoric acid.
Described tumor cell specifically can be lymphoma cell.
When described tumor cell was lymphoma cell: described core sequence can be in the sequence 15 in the sequence table DNA sequence shown in any ten above continuous nucleotides.
When described tumor cell was lymphoma cell: described aptamer was specific as follows:
5 '-CAC CGG GAG GAT AGT TCG GTG GCT GTT CAG GGT CTC CTC CCG GTG-3 ' (sequence 15 of sequence table).
The present invention also protects a kind of drug targeting delivery vehicles, and its active component is described aptamer and/or described nucleic acid aptamer derivative.
The present invention also protects the targeted drug that medicine and the coupling of described drug targeting delivery vehicles are obtained.
The present invention also protects described aptamer and/or the application of described nucleic acid aptamer derivative in drug targeting is carried.
Described medicine can be the medicine of treatment tumor, the medicine that treatment is infected or the medicine for the treatment of inflammation.
The medicine of described treatment tumor specifically can be the kill tumor cell and/or suppresses the medicine of tumor cell proliferation.
Described medicine can be conventional medicine, radioelement, toxin etc.
Described leukaemia specifically can be the CCRF-CEM cell, and lymphoma cell specifically can be the Ramos cell.
The present invention is applied to prepare targeted drug with aptamer and derivant thereof first.At different target cells, can screen different aptamers.With the drug coupling of the fit and existing inhibition target cell of specific nucleic acid, can obtain targeted drug, increase the specificity of medicine to target cell, improve curative effect of medication, reduce drug toxicity, reduce drug dose, thus the toxic and side effects of avoiding.
The screening aptamer specifically can adopt following method: with complete disease cell is target, by combining with at random nucleic acid library, the nucleotide sequence of separating and combining then, after enzyme amplifies, repeat bonded process again, so obtain after the circulation repeatedly multiple can with the bonded aptamer of corresponding disease cell.Usually repeat to screen the 5-20 wheel, can obtain 3-20 energy recognizing cells surface different molecular aptamer, change the nucleic acid recognizing molecule that different cell categories can obtain discerning the various disease cell.Be subjected to the degraded of nuclease in order to resist aptamer, prolong its half-life in organism or biological specimen, before application, aptamer can be carried out structure optimization and/or modification, obtain the derivant of aptamer.
Advantage of the present invention mainly is: can not know to screen under the situation that the molecule of target cell surface with disease association changes and prepare specific aptamer molecule (or nucleic acid aptamer derivative) in advance; Can obtain the aptamer (or nucleic acid aptamer derivative) of various kinds of cell surface disease correlation molecule simultaneously; Resulting aptamer (or nucleic acid aptamer derivative) can combine with the disease cell is powerful by specificity; Aptamer (or nucleic acid aptamer derivative) chemosynthesis on a large scale, be easy to be connected with drug molecule, cost is low; Aptamer (or nucleic acid aptamer derivative) molecular weight is less relatively, does not have immunocompetence and toxicity; Aptamer (or nucleic acid aptamer derivative) good stability, the targeted drug of formation is stable.Targeted drug of the present invention can specificity in conjunction with target cell, obviously reduce poisonous side effect of medicine, improve therapeutic effect, have very high potential using value.
Description of drawings
Fig. 1 is the screening process with the disease association aptamer.
Fig. 2 is aptamer and amycin coupling sketch map
Fig. 3 is the cytoactive analysis result after drug treating.
The specific embodiment
Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique among the following embodiment if no special instructions, is conventional method.Used test material among the following embodiment if no special instructions, is to buy from routine biochemistry reagent company and obtains.
Binding buffer liquid: contain 4.5g/L glucose, 5mM MgCl
2, 1mM CaCl
2, 2.67mM KCl, 1.47mM potassium dihydrogen phosphate, 137.93mM sodium chloride, 8.06mM sodium hydrogen phosphate, 0.1mg/mL tRNA (Sigma) and 1mg/mL bovine serum albumin (Fisher); PH 7.4.
Screening, optimization and the transformation of embodiment 1, aptamer I and aptamer II
(cell A is a target cell to screening process as shown in Figure 1, cellB is anti-sieve cell, 1 expression DNA library at random, 2 expression elution step, 3 expression PCR steps, the library of 4 expression enrichments, 5 expression clone and order-checkings, 6 expressions enter the next round screening, 7 expression flow cytometry steps), specific as follows:
One, the screening of leukaemia CCRF-CEM aptamer, optimization and transformation
(1) screening of aptamer
1, the design in random nucleic acid library is with synthetic
The synthetic two ends of design comprise 18 nucleotide, centres and comprise that the random nucleic acid sequence library of 52 nucleotide is as follows: 5 '-ATA CCA GCT TAT TCA ATT-52-nt-AGA TAG TAA GTG CAA TCT-3 '.
2, the screening of aptamer
As target cell, lymphoma cell Ramos is as anti-sieve cell with leukaemia CCRF-CEM.
(1) first round screening
Get the synthetic random nucleic acid of 5-10nmol step 1 library, be dissolved in the 1mL binding buffer liquid, with 1 * 10
6Individual leukaemia CCRF-CEM (purchases in ATCC, CCL-119) hatched on ice 30 minutes, centrifugal removal supernatant; After the washing with cell suspension in 300 μ L binding buffer liquid and heat the nucleotide sequence of elution of bound in cell surface; The centrifugal cell of removing, with the solution desalination, (primer is right: fluorescein-labeled 5 '-ATA CCA GCT TAT TCA ATT-3 ' through the PCR amplification for the sequence in the solution after the desalination; Biotin labeled 5 '-AGA TTG CAC TTA CTATCT-3 ') back is with the absorption of the agarose microbeads (purchasing the company in GE) of streptavidin bag quilt, obtains single-chain nucleic acid sequence library I through the sodium hydroxide solution eluting of 0.15M, is used for second screening of taking turns.
(2) second take turns screening
Get 200pmol single-chain nucleic acid sequence library I, be dissolved in the 200-300 μ L binding buffer liquid, with 1 * 10
6Individual leukaemia CCRF-CEM was hatched on ice 30 minutes, centrifugal removal supernatant; After the washing with cell suspension in 300 μ L binding buffer liquid and heat the nucleotide sequence of elution of bound in cell surface; Add 5 * 10 again in the centrifugal solution of removing behind the cell
6Individual lymphoma cell Ramos (purchases in ATCC, CRL-1596) in hatching 30 minutes on ice to remove the sequence with the cell non-specific binding; The centrifugal cell of removing, with the solution desalination, sequence in the solution after the desalination is amplified (primer is to same step (1)) back through PCR and is adsorbed with the agarose microbeads (purchasing the company in GE) of streptavidin bag quilt, obtains single-chain nucleic acid sequence library II through the sodium hydroxide solution eluting of 0.15M.
(3) repeat to screen 20 and take turns, method obtains the nucleic acid library that specificity is incorporated into leukaemia CCRF-CEM with step 2.
3, the sign of aptamer molecule
The nucleic acid library that specificity is incorporated into leukaemia CCRF-CEM is cloned and is checked order.Each sequence in library mark fluorescent molecule is respectively made molecular probe, gets 0.5,1,2,4,10,40,200 respectively, the molecular probe solution of 500nM, with 5 * 10
5Individual leukaemia CCRF-CEM is in hatching 20 minutes on ice, with twice back of binding buffer liquid washing cells were tested by flow cytometry cell surface fluorescence intensity, aptamer as cell energy combined with fluorescent labelling, then concentration and probe concentration is mapped, calculate the equilibrium dissociation constant Kd of aptamer with formula Y=BmaxX/ (Kd+X) with fluorescence intensity.
The final aptamer sequence of selecting is as follows:
sgc3：5’-ATACCAGCTTATTCAATT
CCTGTGGGAAGGCTATAGAGGGGCCAGTCTATGAAT AAGATGGCGGACTAATGTGTAAGATAGTAAGTGCAATCT-3’；
What wherein add the thick underline labelling is core sequence, the sequence 1 of seeing sequence table.
sgc6：5’-ATACCAGCTTATTCAATT
CCTGTGGGAAGGCTATAGAGGGGCCAGTCTATGAAC AAGATGGTTGATCCGTAGATAGTAAGTGCAATCT-3’；
What wherein add the thick underline labelling is core sequence, the sequence 2 of seeing sequence table.
sgd3：5’-ATACCAGCTTATTCAATT
AGGGGGAGCTTGCGCGCATCAAGGTGCTAAACGAAA GCCTCATGGCTTCTATAGATAGTAAGTGCAATCT-3’；
What wherein add the thick underline labelling is core sequence, the sequence 3 of seeing sequence table.
sgc4：5’-ATACCAGCTTATTCAATT
CGAGTGCGGATGCAAACGCCAGACAGGGGGACAGGA GATAAGAATAGCGTGATGAGATAGTAAGTGCAATCT-3’；
What wherein add the thick underline labelling is core sequence, the sequence 4 of seeing sequence table.
sgc4a：5’-ATACCAGCTTATTCAATT
CGAGTGCGGATGCAAACGCCAGACAGGGGGACAGGAGATAGTAAGTGCAATCT-3’；
What wherein add the thick underline labelling is core sequence, the sequence 5 of seeing sequence table.
sgc5：5’-ATACCAGCTTATTCAATT
ACCGACGACGAACTATCTATCACTATCTTACACATC ATACCTCGAGATAGTAAGTGCAATCT-3’；
What wherein add the thick underline labelling is core sequence, the sequence 6 of seeing sequence table.
sgc7：5’-ATACCAGCTTATTCAATT
ACCGCAGCGACTATCTCGACTACATTACTAGCTTATA CTCCGATCATCTCTAGATAGTAAGTGCAATCT-3’；
What wherein add the thick underline labelling is core sequence, the sequence 7 of seeing sequence table.
sgc8：5’-ATACCAGCTTATTCAATT
AGTCACACTTAGAGTTCTAACTGCTGCGCCGCCGGGA AAATACTGTACGGTTAGATAGTAAGTGCAATCT-3’；
What wherein add the thick underline labelling is core sequence, the sequence 8 of seeing sequence table.
sga16：5’-ATACCAGCTTATTCAATT
AGTCACACTTAGAGTTCTAGCTGCTGCGCCGCCGGG AAAATACTGTACGGATAGATAGTAAGTGCAATCT-3’；
What wherein add the thick underline labelling is core sequence, the sequence 9 of seeing sequence table.
Sgd2：5’-ATACCAGCTTATTCAATT
GAGTGAAGCAAGGATGCAACCTCGGCTCCAACCCGT GAGAGTCGCGAAACTCAGATAGTAAGTGCAATCT-3’；
What wherein add the thick underline labelling is core sequence, the sequence 10 of seeing sequence table.
Sgd5：5’-ATACCAGCTTATTCAATT
ATCGTGGGTCACAGCAGCGGTTGTGAGGAAGAAAGGC GGATAACAGATAATAAGATAGTAAGTGCAATCT-3’；
What wherein add the thick underline labelling is core sequence, the sequence 11 of seeing sequence table.
(2) optimization of aptamer molecule and transformation
The aptamer that step 1 obtains is longer, after structural analysis, design and synthesize a series of nucleotide sequences through truncate, characterize the specificity of sequence and affinity (the same step of method () 3) with flow cytometer, select optimized binding sequence and be used for further application, the length of the sequence after the optimization can be reduced to 1/3rd of former sequence.The sequence (sgc8c) (see the sequence 14 of sequence table) of aptamer sgc8 after optimizing is as follows:
5’-A
TC?TAA?CTG?CTG?CGC?CGC?CGG?GAA?AAT?ACT?GTA?CGG?TTA?GA-3’；
The core sequence that is that wherein adds the thick underline labelling.
Sgc8c selects the good short-movie section of adhesion from sgc8, the A (also can be other any nucleotide) that is used for labelling in its 5 ' one of terminal interpolation obtains.
4 nucleotide of the 5 ' end of sequence sgc8c and 3 nucleotide of 3 ' end are replaced with corresponding LNA (lock nucleic acid) unit (A
L, C
L, T
L, G
L), will be wherein sequence A AAATACT replace with two six Polyethylene Glycol-1-phosphoric acid link molecule (monomer of the synthetic usefulness of DNA is: Spacer Phosphoramidite 18,
18-O-Dimethoxytritylhexaethyleneglycol, 1-[(2-cyanoethyl)-(N, N-diisopropyl)]-phosphoramidite, 10-1918-02, Glen Research) sequence A that (peg) must modify
LT
LC
LT
LAACTGCTGCGCCGCCGGGpegGTACGGTTA
LG
LA
L, with its called after aptamer A.The half-life of sequence in blood plasma before modifying is 1.5 hours, is 8 hours after the modification.
Two, the screening of lymphoma cell Ramos aptamer, optimization and transformation
(1) screening of aptamer
1, the design in random nucleic acid library is with synthetic
The synthetic two ends of design comprise 20 nucleotide, centres and comprise that the random nucleic acid sequence library of 45 nucleotide is as follows: 5 '-AAG GAG CAG CGT GGA GGA TA-N45-TTA GGG TGT GTC GTC GTG GT-3 '.
2, the screening of aptamer
As target cell, making leukaemia CCRF-CEM is anti-sieve cell with lymphoma cell Ramos.
Other is with (one's) of step 12.
3, the sign of aptamer molecule
With (one) of step 13.
(2) optimization of aptamer molecule and transformation
(two) with step 1.
The aptamer sequence of the optimization that finally obtains is as follows:
TD05:5 '-CAC CGG GAG GAT AGT TCG GTG GCT GTT CAG GGT CTC CTC CCG GTG-3 ' (seeing the sequence 15 of sequence table).
The preparation of embodiment 2, targeted drug and application
One, the preparation of targeted drug I
Preparation flow as shown in Figure 2.
1, the preparation of amycin 6-maleimide hexanoyl hydrazone
Get doxorubicin hydrochloride (5mg, 8.62 μ mol) and EMCH (N-e-(Maleimidocaproic acid) hydrazide, N-e-maleimide hexanoyl hydrazine) (Pierce Biotechnology) (10mg, 44.4 μ mol) be dissolved in 4mL methanol, add 3 μ L trifluoroacetic acids in room temperature lucifuge reaction 24 hours; Room temperature is evaporated to 0.25mL then, adds the 2.5mL acetonitrile and places 48 hours recrystallization in 4 ℃; With methanol-acetonitrile (1: 10) mixed liquor washing crystal, vacuum drying gets amycin 6-maleimide hexanoyl hydrazone (2.9mg, 3.85 μ mol).
2, the coupling of aptamer and medicine
The aptamer A that gets 300nmol 5 ' end disulfide bond modification is dissolved in the PBS buffer (pH 7.4), at room temperature with TCEP (Tris (2-carboxyethyl) phosphine, three (2-carbonyl ethyl) phosphorus, 20490, Thermo Fisher Scientific Inc.) react 2 hours with the reduction disulfide bond.By Sephadex G-25 post (NAP
TM-5, Amersham Pharmacia Biotech) removes behind the excessive TCEP with 2 μ mol amycin 6-maleimide hexanoyl hydrazones in 50 μ L DMF (dimethyl fumarate) in reacting 12 hours on ice.Product is targeted drug I with high-efficient liquid phase chromatogram purification (140nmol).Among the targeted drug I, the mol ratio of aptamer A and amycin is 1: 1.
Two, the preparation of targeted drug II
1, the preparation of amycin 6-maleimide hexanoyl hydrazone
With 1 of step 1
2, the coupling of aptamer and medicine
Get the TD05 sequence that 300nmol 5 ' end disulfide bond is modified, be dissolved in the PBS buffer (pH 7.4), at room temperature react 2 hours with the reduction disulfide bond with TCEP.By Sephadex G-25 post (NAP
TM-5, AmershamPharmacia Biotech) removes behind the excessive TCEP with 2 μ mol amycin 6-maleimide hexanoyl hydrazones in 50 μ L DMF in reacting 12 hours on ice.Product is targeted drug II with high-efficient liquid phase chromatogram purification (140nmol).Among the targeted drug II, the mol ratio of TD05 sequence and amycin is 1: 1.
Three, targeted drug is to the cytotoxicity analysis of CCRF-CEM
Cytotoxicity analysis carries out in 96 orifice plates, and 2 * 10
4Individual leukaemia CCRF-CEM/ hole.Four groups of processing are set, and each processing is provided with three repetitions:
Handle one: add amycin, the amount of amycin is respectively 0 to 2.5 μ M in every hole;
Handle two: add targeted drug I, the amount of amycin is respectively 0 to 2.5 μ M in every hole;
Handle three: add targeted drug II, the amount of amycin is respectively 0 to 2.5 μ M in every hole;
Handle four: add aptamer A, the amount of the fit A of every hole amplifying nucleic acid is respectively 0 to 2.5 μ M.
With untreated cell in contrast.Hatch after two hours and continue to cultivate 48 hours after the culture medium with fresh culture displacement 75%.Adding 20 μ L CellTiter reagent in every hole (Promega, Madison, WI).Hatch the absorption of measuring 490nm after 2 hours with microplate reader.With the trap value in the hole that contains unprocessed cell is 100%, the trap in cytoactive=the contain hole of different disposal cell/contain trap * 100% in the hole of unprocessed cell.
The cell activity analysis result as shown in Figure 3 after the different disposal.
Targeted drug II and aptamer A do not see obvious cytotoxicity, cytotoxic drug coupling nucleic acid sequence is described after because aqueous solution increases and can not freely diffuse into cell, so the non-specific cell cytotoxic activity of conjugate reduces greatly.The cytotoxic activity of targeted drug I is similar to amycin, illustrates that aptamer has the function of targeting delivery medicine, and targeted drug can deliver medicine with the corresponding internalization process that causes after cell combines by aptamer and enter cell.
Four, targeted drug is to the oxicity analysis of Ramos
Cytotoxicity analysis carries out in 96 orifice plates, and 2 * 10
4Individual lymphoma cell Ramos/ hole.Four groups of processing are set, and each processing is provided with three repetitions:
Handle one: add amycin, the amount of amycin is respectively 0 to 2.5 μ M in every hole;
Handle two: add targeted drug I, the amount of amycin is respectively 0 to 2.5 μ M in every hole;
Handle three: add targeted drug II, the amount of amycin is respectively 0 to 2.5 μ M in every hole;
Handle four: add aptamer A, the amount of the fit A of every hole amplifying nucleic acid is respectively 0 to 2.5 μ M.
With untreated cell in contrast.Hatch after two hours and continue to cultivate 48 hours after the culture medium with fresh culture displacement 75%.Adding 20 μ L CellTiter reagent in every hole (Promega, Madison, WI).Hatch the absorption of measuring 490nm after 2 hours with microplate reader.Trap value with the hole that contains unprocessed cell is 100%, the trap in cytoactive=the contain hole of different disposal cell/contain hole trap * 100% of unprocessed cell.
The result shows: aptamer A, targeted drug I there is no obvious cytotoxicity, cytotoxic drug coupling nucleic acid sequence is described after because aqueous solution increases and can not freely diffuse into cell, so the non-specific cell cytotoxic activity of conjugate reduces greatly.And amycin has similar cytotoxic activity to targeted drug II, but because therefore the aptamer specific recognition Ramos cell among the targeted drug II causes the targeted cells cytotoxic activity, proves absolutely the targeting delivery function of aptamer.
Sequence table
＜110〉Tan Weihong
＜120〉the new purposes of aptamer and derivant thereof
<130>CGGNAY92235
<160>15
<210>1
<211>57
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>1
cctgtgggaa?ggctatagag?gggccagtct?atgaataaga?tggcggacta?atgtgta 57
<210>2
<211>52
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>2
cctgtgggaa?ggctatagag?gggccagtct?atgaacaaga?tggttgatcc?gt 52
<210>3
<211>52
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>3
agggggagct?tgcgcgcatc?aaggtgctaa?acgaaagcct?catggcttct?at 52
<210>4
<211>54
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>4
cgagtgcgga?tgcaaacgcc?agacaggggg?acaggagata?agaatagcgt?gatg 54
<210>5
<211>35
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>5
cgagtgcgga?tgcaaacgcc?agacaggggg?acagg 35
<210>6
<211>44
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>6
accgacgacg?aactatctat?cactatctta?cacatcatac?ctcg 44
<210>7
<211>51
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>7
accgcagcga?ctatctcgac?tacattacta?gcttatactc?cgatcatctc?t 51
<210>8
<211>52
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>8
agtcacactt?agagttctaa?ctgctgcgcc?gccgggaaaa?tactgtacgg?tt 52
<210>9
<211>52
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>9
agtcacactt?agagttctag?ctgctgcgcc?gccgggaaaa?tactgtacgg?at 52
<210>10
<211>52
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>10
gagtgaagca?aggatgcaac?ctcggctcca?acccgtgaga?gtcgcgaaac?tc 52
<210>11
<211>52
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>11
atcgtgggtc?acagcagcgg?ttgtgaggaa?gaaaggcgga?taacagataa?ta 52
<210>12
<211>18
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>12
ataccagctt?attcaatt 18
<210>13
<211>18
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>13
agatagtaag?tgcaatct 18
<210>14
<211>41
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)
＜223〉n=a or c or g or t
<400>14
ntctaactgc?tgcgccgccg?ggaaaatact?gtacggttag?a 41
<210>15
<211>45
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>15
caccgggagg?atagttcggt?ggctgttcag?ggtctcctcc?cggtg 45
Claims (10)
1, aptamer and/or the nucleic acid aptamer derivative application in the preparation targeted drug;
Described nucleic acid aptamer derivative be following a), b) or c):
A) skeleton with aptamer partly or entirely transform phosphorothioate backbone as or the sugar ring on the part or all of skeleton is carried out 2 '-F and 2 '-NH
2Modify and wait the nucleic acid aptamer derivative that obtains;
B) partly or entirely transform aptamer as nucleic acid aptamer derivative that peptide nucleic acid(PNA) obtains;
C) an above nucleotide in the aptamer is replaced with the nucleic acid aptamer derivative that lock nucleic acid and/or link molecule obtain; Described link molecule is more than one six Polyethylene Glycol-1-phosphoric acid.
2, application as claimed in claim 1 is characterized in that: described targeted drug is the medicine of treatment tumor, the medicine that treatment is infected or the medicine for the treatment of inflammation; The medicine of described treatment tumor is kill tumor cell and/or the medicine that suppresses tumor cell proliferation.
3, application as claimed in claim 2 is characterized in that: described tumor cell is the leukaemia; Described aptamer is the DNA that contains core sequence; Described core sequence can be in sequence 1, sequence 2, sequence 3, sequence 4, sequence 5, sequence 6, sequence 7, sequence 8, sequence 9, sequence 10 or the sequence 11 in the sequence table DNA sequence shown in any ten above continuous nucleotides.
4, application as claimed in claim 3 is characterized in that: described aptamer is shown in the sequence 14 of sequence table; Described nucleic acid aptamer derivative will replace with the nucleic acid aptamer derivative that described link molecule obtains from 5 ' terminal the 23rd to 30 nucleotide for 4 nucleotide of 5 ' end of the aptamer shown in the sequence 14 of sequence table and 3 nucleotide of 3 ' end are replaced with corresponding lock nucleic acid unit; Described link molecule is two six Polyethylene Glycol-1-phosphoric acid.
5, application as claimed in claim 2 is characterized in that: described tumor cell is a lymphoma cell; Described aptamer is the DNA that contains the sequence 15 of ordered list.
6, a kind of drug targeting delivery vehicles, its active component is aptamer and/or nucleic acid aptamer derivative;
Described nucleic acid aptamer derivative be following a), b) or c):
A) skeleton with aptamer partly or entirely transform phosphorothioate backbone as or the sugar ring on the part or all of skeleton is carried out 2 '-F and 2 '-NH
2Modify and wait the nucleic acid aptamer derivative that obtains;
B) partly or entirely transform aptamer as nucleic acid aptamer derivative that peptide nucleic acid(PNA) obtains;
C) an above nucleotide in the aptamer is replaced with the nucleic acid aptamer derivative that lock nucleic acid and/or link molecule obtain; Described link molecule is more than one six Polyethylene Glycol-1-phosphoric acid.
7, drug targeting delivery vehicles as claimed in claim 6 is characterized in that: described medicine is the medicine of treatment tumor, the medicine that treatment is infected or the medicine for the treatment of inflammation; The medicine of described treatment tumor is kill tumor cell and/or the medicine that suppresses tumor cell proliferation.
8, drug targeting delivery vehicles as claimed in claim 7 is characterized in that: be following 1) or 2):
1) described tumor cell is the leukaemia; Described nucleic acid aptamer derivative will replace with the nucleic acid aptamer derivative that described link molecule obtains from 5 ' terminal the 23rd to 30 nucleotide for 4 nucleotide of 5 ' end of the aptamer shown in the sequence 14 of sequence table and 3 nucleotide of 3 ' end are replaced with corresponding lock nucleic acid unit; Described link molecule is two six Polyethylene Glycol-1-phosphoric acid;
2) described tumor cell is a lymphoma cell; Described aptamer is the DNA that contains the sequence 15 of ordered list.
9, a kind of targeted drug obtains arbitrary described drug targeting delivery vehicles coupling in medicine and the claim 6 to 8.
10, aptamer and/or the nucleic acid aptamer derivative application in drug targeting is carried;
Described nucleic acid aptamer derivative be following a), b) or c):
A) skeleton with aptamer partly or entirely transform phosphorothioate backbone as or the sugar ring on the part or all of skeleton is carried out 2 '-F and 2 '-NH
2Modify and wait the nucleic acid aptamer derivative that obtains;
B) partly or entirely transform aptamer as nucleic acid aptamer derivative that peptide nucleic acid(PNA) obtains;
C) an above nucleotide in the aptamer is replaced with the nucleic acid aptamer derivative that lock nucleic acid and/or link molecule obtain; Described link molecule is more than one six Polyethylene Glycol-1-phosphoric acid.
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| CN2009100830800A CN101537189B (en) | 2009-04-28 | 2009-04-28 | Aptamer and new use of derivative thereof |
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| CN2009100830800A CN101537189B (en) | 2009-04-28 | 2009-04-28 | Aptamer and new use of derivative thereof |
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| CN101537189B CN101537189B (en) | 2013-07-31 |
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